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Role of Biofuels in Energy Transition, Green Economy andCarbon Neutrality

Nida Khan 1, Kumarasamy Sudhakar 1,2,3,* and Rizalman Mamat 4

��

Citation: Khan, N.; Sudhakar, K.;

Mamat, R. Role of Biofuels in Energy

Transition, Green Economy and

Carbon Neutrality. Sustainability 2021,

13, 12374. https://doi.org/10.3390/

su132212374

Academic Editor: Paris Fokaides

Received: 28 July 2021

Accepted: 28 October 2021

Published: 9 November 2021

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1 Faculty of Mechanical and Automotive Engineering Technology, Universiti Malaysia Pahang,Pekan 26600, Malaysia; [email protected]

2 Automotive Engineering Centre, Universiti Malaysia Pahang, Pekan 26600, Malaysia3 Energy Centre, Maulana Azad National Institute of Technology, Bhopal 462003, India4 School of Mechanical Engineering, Ningxia University, Yinchuan 750021, China; [email protected]* Correspondence: [email protected]

Abstract: Modern civilization is heavily reliant on petroleum-based fuels to meet the energy demandof the transportation sector. However, burning fossil fuels in engines emits greenhouse gas emis-sions that harm the environment. Biofuels are commonly regarded as an alternative for sustainabletransportation and economic development. Algal-based fuels, solar fuels, e-fuels, and CO2-to-fuelsare marketed as next-generation sources that address the shortcomings of first-generation andsecond-generation biofuels. This article investigates the benefits, limitations, and trends in differentgenerations of biofuels through a review of the literature. The study also addresses the newer genera-tion of biofuels highlighting the social, economic, and environmental aspects, providing the readerwith information on long-term sustainability. The use of nanoparticles in the commercialization ofbiofuel is also highlighted. Finally, the paper discusses the recent advancements that potentiallyenable a sustainable energy transition, green economy, and carbon neutrality in the biofuel sector.

Keywords: biofuels; sustainability; bioeconomy; solar fuels

1. Introduction

Our planet is experiencing more natural calamities that are severe in terms of intensityand duration. The use of non-renewable fuels as primary energy sources for severalyears resulted in increasing the speed of global warming and the emission of various airpollutants that are detrimental to the environment and public health. According to a reviewof five leading international datasets by the World Meteorological Organization (WMO),2020 was one of the three warmest years on earth, tied with 2016 for first place [1]: anotherstark reminder of the accelerated pace of climate change, which is devastating health andlives around our world. Based on current policies, global energy demand is expected torise by 1.3% per year until 2040 without dramatic energy production and recycling [2].Progress must be made even sooner to reduce greenhouse gas associated with industrialdevelopment and energy usage. By 2070, the International Energy Agency (IEA) anticipatesglobal transportation (measured in passenger kilometers) to be increased fourfold and carownership rates to rise by 60%. According to the Energy Technology Perspective study, thedemand for passenger and freight aircraft will triple [3].

The idea of using biofuels appears to be feasible to bring our planet on the pathway tomeet energy-related sustainable development. Henry Ford (1896) pioneered bioethanol,while Rudolf Diesel was an innovator in peanut oil. Biofuel is one of the sustainable energysources obtained from processing various feedstocks such as plant, algae, or animal waste.Biodiesel (fatty acid methyl ester, or FAME, fuels derived from vegetable oils and fats,including wastes such as used cooking oil) and bioethanol (produced from corn, sugarcane, and other crops) are the two most popular biofuels. Since liquid fossil fuels dominatethe transportation sector, replacing these fuels with renewable energy will significantly

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contribute to the achievement of the comprehensive energy and sustainability goals. Themost widely used biofuels are ethanol (from various sources), which is well suited toOtto cycle engines, and biodiesel (from multiple sources), which is better suited to dieselcycle engines. Biodiesel can be used as a fuel additive in compression ignition (diesel)engines, primarily in 20% blends (B20) with petroleum diesel. Biodiesel blend levelsare often determined by the cost of the fuel and the projected advantages. Biomethanefuels in CNG buses demonstrate the sustainability concept while also improving overallenvironmental performance.

Several countries have now passed regulations approving biofuels to meet the po-tential transportation requirements [4]. The integration of biofuels will reduce a nation’sreliance on conventional petroleum imports from other countries, which will help mitigatethe impacts of the fluctuations in oil prices, boost the economy, and reduce carbon emis-sions. Moreover, biofuels encourage new entrepreneurs while simultaneously increasingeconomic activity globally. They also provide community-level growth alternatives forsmall and medium-size power grids [5].

Overall, global ethanol production decreased about 15% in 2020 and biodiesel pro-duction decreased by 5% in 2020. The International Energy Agency (IEA) estimates thatworldwide transportation biofuel output will return to approximately 162 billion litersin 2021, similar to 2019 [6]. In 2025, biofuels are expected to provide roughly 5.4% of theenergy requirement for road transport [7].

Dependent on feedstocks and technique, biofuels are grouped into multiple categoriesknown as 1st, 2nd, 3rd, and 4th generation. Agricultural products or traditional biofuels areused to generate first-generation biofuels. Fermentation, transesterification, and anaerobicdigestion are examples of comparatively well-established processes for producing thesefuels [8]. The primary concern with first-generation biofuel is that it is primarily made fromagricultural resources, which has a negative impact on financial, ecological, and politicalconsiderations because mass biofuel production necessitates more fertile land, resultingin far fewer lands available for human and animal food production [9]. Lignocellulosicfeedstocks, agro-residues, and non-edible plant biomass constitute the second-generationfeedstocks [10]. Biofuels of the second generation overcome the impact on the climate andsocial aspects. However, it has a negative energy yield, feedstock transportation issues,high downstream production costs, and modest greenhouse gas (GHG) reduction, limitingtheir use [11].

On the other hand, biofuels of the 3rd generation have gained broad interest as asubstitute for biofuel production to address the problems associated with the first andsecond generations [8]. The most promising feedstock for renewable fuel production ismacro and microalgae. Microalgae and macroalgae require sunlight [12], water, nutrients,and carbon dioxide to create energy biofuels. Algae biomass has the distinct benefits of notcompeting with soil, having low lignin content, requiring less energy, and competing lesswith food crops [13]. Genetically modified (GM) algae are used in fourth-generation biofuel(FGB), but there is still considerable concern about the negative environmental impacts.

Biofuels for Transportation

Figure 1 depicts non-renewable biofuels used in the different transportation sectors.Biomethane is utilized to fuel CNG buses, demonstrating that the gas used to powerCNG buses may be produced sustainably while also increasing overall environmentalperformance. The most promising bio-derived fuels for ship use are SVO and biodiesel.The importance of marine transportation in global freight distribution cannot be overstated.However, the maritime sector accounts for nearly 2.6% of worldwide GHG emissions.ExxonMobil has completed a successful sea trial of its first marine biofuel oil with StenaBulk, which is a shipping firm bunkered in Rotterdam. The study showed that marinebiofuel oil, which may reduce CO2 emissions by up to 40% when compared to traditionalmarine fuel, can be utilized in a relevant maritime application without modification,allowing operators to make substantial progress toward their carbon reduction goals. The


aviation industry is responsible for 12% of all transportation-related GHG emissions and2–3% of all anthropogenic GHG emissions [12]. Hundreds of demonstration flights havebeen flown by more than 20 airlines using a combination of regular jet fuel and aviation-grade biofuel generated from various feedstocks, including waste cooking oil and oil cropssuch as rapeseed, jatropha, camelina, and palm oil, to produce an alternate aviation biofuel.

Sustainability 2021, 13, x FOR PEER REVIEW 3 of 32

Bulk, which is a shipping firm bunkered in Rotterdam. The study showed that marine biofuel oil, which may reduce CO2 emissions by up to 40% when compared to traditional marine fuel, can be utilized in a relevant maritime application without modification, al-lowing operators to make substantial progress toward their carbon reduction goals. The aviation industry is responsible for 12% of all transportation-related GHG emissions and 2–3% of all anthropogenic GHG emissions [12]. Hundreds of demonstration flights have been flown by more than 20 airlines using a combination of regular jet fuel and aviation-grade biofuel generated from various feedstocks, including waste cooking oil and oil crops such as rapeseed, jatropha, camelina, and palm oil, to produce an alternate aviation biofuel.

Figure 1. Biofuels as an alternative for non-renewable fuels in the different transportation sectors.

The purpose of this article is to shed light on the following aspects of biofuel: (a) To investigate the benefits, limitations, and trends in different generations of biofu-

els. (b) To assess the social, economic, and environmental effects for the long-term sustaina-

bility of biofuels. (c) To highlight the recent advancements in the biofuel sector that potentially enable

carbon neutrality, sustainable energy transition, and a greener economy.

2. Overview of 1G, 2G, 3G, and 4G Generations of Biofuels Figure 2 depicts different generations of biofuels based on the feedstock and the de-

velopment of the conversion process.

Light Vehicles

- Bioethanol

- Biodiesel

Heavy Vehicles

- Biodiesel

Heavy Machinery- Biodiesel

Marine- Bioethanol

-Biodiesel

Aviation- Sustainable Aviation Fuel

Figure 1. Biofuels as an alternative for non-renewable fuels in the different transportation sectors.

The purpose of this article is to shed light on the following aspects of biofuel:

(a) To investigate the benefits, limitations, and trends in different generations of biofuels.(b) To assess the social, economic, and environmental effects for the long-term sustain-

ability of biofuels.(c) To highlight the recent advancements in the biofuel sector that potentially enable

carbon neutrality, sustainable energy transition, and a greener economy.

2. Overview of 1G, 2G, 3G, and 4G Generations of Biofuels

Figure 2 depicts different generations of biofuels based on the feedstock and thedevelopment of the conversion process.


Figure 2. Biofuels generation.

2.1. First Generation (1G) First-generation biofuels include biodiesel, bioethanol, and biogas, which are used

commercially. Biodiesel is a diesel substitute produced by the oil transesterification of natural sources as well as leftover fats and oils. At the same time, bioethanol is a gasoline substitute that is produced through the fermentation of sugar or starch as illustrated in Figure 3. First-generation biofuels are being evaluated based on two main claims: For in-stance, they explicitly attempt to compete with crops for feed. Second, their energy, eco-nomic, and environmental balance will not be as optimal as previously planned. Accord-ing to many researchers, if food prices are influenced by biofuel production to the same extent, the number of food-insecure people in developed countries will increase to nearly 1.2 billion by 2025 [13].

Figure 3. First-generation biofuels.

Several studies have found that switching to first-generation biofuels may result in an increase in GHG emissions. Senauer [13] stated that agricultural use and fertilizer ap-plication will double emissions over the next 30 years rather than the anticipated 20% reduction in GHG emissions from biofuel. Furthermore, 1G biofuels such as ethanol re-quire a large amount of maize, which requires a large amount of water ranging from 5 to 2138 liters (L) per 1 L of ethanol, depending on how and where ethanol maize is grown. [14]. This appears to have negative environmental consequences, as its intake from water sources can put those areas at risk of drought. Pursuing biofuel production in water-scarce


2.1. First Generation (1G)

First-generation biofuels include biodiesel, bioethanol, and biogas, which are usedcommercially. Biodiesel is a diesel substitute produced by the oil transesterification of


natural sources as well as leftover fats and oils. At the same time, bioethanol is a gasolinesubstitute that is produced through the fermentation of sugar or starch as illustrated inFigure 3. First-generation biofuels are being evaluated based on two main claims: Forinstance, they explicitly attempt to compete with crops for feed. Second, their energy,economic, and environmental balance will not be as optimal as previously planned. Ac-cording to many researchers, if food prices are influenced by biofuel production to thesame extent, the number of food-insecure people in developed countries will increase tonearly 1.2 billion by 2025 [13].



2.1. First Generation (1G) First-generation biofuels include biodiesel, bioethanol, and biogas, which are used

commercially. Biodiesel is a diesel substitute produced by the oil transesterification of natural sources as well as leftover fats and oils. At the same time, bioethanol is a gasoline substitute that is produced through the fermentation of sugar or starch as illustrated in Figure 3. First-generation biofuels are being evaluated based on two main claims: For in-stance, they explicitly attempt to compete with crops for feed. Second, their energy, eco-nomic, and environmental balance will not be as optimal as previously planned. Accord-ing to many researchers, if food prices are influenced by biofuel production to the same extent, the number of food-insecure people in developed countries will increase to nearly 1.2 billion by 2025 [13].


Several studies have found that switching to first-generation biofuels may result in an increase in GHG emissions. Senauer [13] stated that agricultural use and fertilizer ap-plication will double emissions over the next 30 years rather than the anticipated 20% reduction in GHG emissions from biofuel. Furthermore, 1G biofuels such as ethanol re-quire a large amount of maize, which requires a large amount of water ranging from 5 to 2138 liters (L) per 1 L of ethanol, depending on how and where ethanol maize is grown. [14]. This appears to have negative environmental consequences, as its intake from water sources can put those areas at risk of drought. Pursuing biofuel production in water-scarce


Several studies have found that switching to first-generation biofuels may result inan increase in GHG emissions. Senauer [13] stated that agricultural use and fertilizerapplication will double emissions over the next 30 years rather than the anticipated 20%reduction in GHG emissions from biofuel. Furthermore, 1G biofuels such as ethanolrequire a large amount of maize, which requires a large amount of water ranging from 5 to2138 liters (L) per 1 L of ethanol, depending on how and where ethanol maize is grown. [14].This appears to have negative environmental consequences, as its intake from water sourcescan put those areas at risk of drought. Pursuing biofuel production in water-scarce locationswould further strain an already constrained resource, mainly if a crop requires irrigation.Water resources and wetlands are expected to suffer as a result of increased water intake [15].Chaudhary et al. [16] examined the ecological impacts of ethanol production in variousparts of the world. It was demonstrated that the cultivation of sugar cane in Brazil suffers agreater loss of biodiversity than the production of sugar beet in France and maize (grain orstover) in the United States [16]. The expansion of 1G biofuels has been a source of socialstress, particularly in developing countries where biofuel expansion has occurred in theabsence of advanced facilities to control it. Biofuel-based community conflicts are typicallyrelated to land contract issues. Citizens in Tanzania, Mozambique, Ghana, Kenya, andZambia have been reported to have lost access to their shared land due to extensive jatrophafarming. Land leases are frequently at the core of biofuel-related community disputes [17].The Indian government’s and the biofuel industry’s rapid adoption of jatropha threatensto drive millions of underprivileged rural farmers out of areas where they get their food,fuel, wood, fodder, and lumber [18]. Conventional agricultural production is alreadyfacing extreme water constraints; therefore, the regional and local water supply burdenwould be enormous with 1G biofuel. Policymakers would be hesitant to pursue biofuelalternatives based on conventional food and oil crops [19].The pros and cons of the 1Gbiofuels are highlighted in Figure 4. The biofuel yield parameters from 1G feedstock isprovided in Table 1.



locations would further strain an already constrained resource, mainly if a crop requires irrigation. Water resources and wetlands are expected to suffer as a result of increased water intake [15]. Chaudhary et al. [16] examined the ecological impacts of ethanol pro-duction in various parts of the world. It was demonstrated that the cultivation of sugar cane in Brazil suffers a greater loss of biodiversity than the production of sugar beet in France and maize (grain or stover) in the United States [16]. The expansion of 1G biofuels has been a source of social stress, particularly in developing countries where biofuel ex-pansion has occurred in the absence of advanced facilities to control it. Biofuel-based com-munity conflicts are typically related to land contract issues. Citizens in Tanzania, Mozambique, Ghana, Kenya, and Zambia have been reported to have lost access to their shared land due to extensive jatropha farming. Land leases are frequently at the core of biofuel-related community disputes [17]. The Indian government’s and the biofuel indus-try’s rapid adoption of jatropha threatens to drive millions of underprivileged rural farm-ers out of areas where they get their food, fuel, wood, fodder, and lumber [18]. Conven-tional agricultural production is already facing extreme water constraints; therefore, the regional and local water supply burden would be enormous with 1G biofuel. Policymak-ers would be hesitant to pursue biofuel alternatives based on conventional food and oil crops [19].The pros and cons of the 1G biofuels are highlighted in Figure 4. The biofuel yield parameters from 1G feedstock is provided in Table 1.

Figure 4. Pros and cons of first-generation biofuels.

+ -

Figure 4. Pros and cons of first-generation biofuels.

Table 1. Parameters and yield of biodiesel, biomethane, bioethanol, and syngas for various feedstocks of first-generation biofuels.

Biofuels Biodiesel Bioethanol Biomethane Biobutanol Syngas

Feedstock Soybean Corn Corn Corn Rapeseed

Parameters

Transesterification,Hydrotalcite as basic

catalyst,methanol/oil molar

ratio of 20:1,reaction time of 10 h

Fermentation,Primary liquefaction,

heat treatment(105–110 ◦C)for 5–7 min,

α-amylase Secondaryliquefaction: 95 ◦C,1–2 h, enzymatic

liquefaction,pH 4.5

Anaerobic digestion,pH-range—6.5–8.2.

mesophilic(30–40 ◦C),

or thermophilic(50–60 ◦C)

AB Fermentation,Strain: C. acetobutylicum,Temperature 34 to 39 ◦C

for 40 to 60 h

Fast pyrolysis,Conversion to

biocharSteam gasification

Steam flow:172 g min−1 kg−1

biocharTemperature = 750 ◦C

Yield 189.6 g/kg 449 g/kg 205–450 dm3kg−1 12–20 g/L H2 = 58.7%CO = 10.6%

References [20] [21] [22] [23] [24]

2.2. Second Generation (2G)

Figure 5 provides the 2G biofuels feedstock. Second-generation biofuels addressmany of the issues related to first-generation biofuels. The prospects for fostering regionalgrowth and improving the economic situation in developing regions is envisaged with 2Gbiofuels. Around the world, various strategies for the production of second-generationbiofuels are being considered. Still, the focus is primarily on two distinct paths, eitherthe thermo or bio route generated by biomass of cellulose and lignin, tree surplus, and


seasonal forage crop. Thermochemical manufacturing has the significant benefit of higherversatility of feedstock than biological production. The “thermo” route is focused onthe heat processing of biomass under decreased oxidizing agent concentrations. Underthe temperature range of 300 to 1000 ◦C, the bottom range will primarily produce solidbiofuel called biochar. At elevated temperatures, pyrolytic oil and syngas are the mostconcentrated substances in the middle range. The “bio” approach includes the pretreatmentof lignocellulosic material, enzymatic hydrolysis, and the fermentation of sugars by specificstrains of microorganism. It is more challenging to convert lignocellulose to reducingsugars than it is to convert starch. Biological, physical (thermal), or chemical catalystsare used to pretreat biomass in the biochemical pathway as shown in Figure 6; hence,enhanced advances in the development of 2G biofuels are hampered due to the chemicaland structural properties of the extracellular matrix. The biodiesel yield parameters fromthe 2G biofuel are highlighted in Table 2.


Table 1. Parameters and yield of biodiesel, biomethane, bioethanol, and syngas for various feedstocks of first-generation biofuels.

Biofuels Biodiesel Bioethanol Biomethane Biobutanol Syngas Feedstock Soybean Corn Corn Corn Rapeseed

Parameters

Transesterification, Hydrotalcite as basic

catalyst, methanol/oil molar

ratio of 20:1, reaction time of 10 h

Fermentation, Primary liquefaction,

heat treatment (105–110 °C) for 5–7 min,

α-amylase Secondary liquefaction: 95 °C,

1–2 h, enzymatic liquefaction, pH 4.5

Anaerobic digestion, pH-range—6.5–8.2.

mesophilic (30–40 °C), or thermophilic (50–60 °C)

AB Fermentation, Strain: C. acetobutylicum, Temperature 34 to 39 °C

for 40 to 60 h

Fast pyrolysis, Conversion to biochar

Steam gasification Steam flow:

172 gmin_1 kg_1 biochar Temperature = 750 °C

Yield 189.6 g/kg 449 g/kg 205–450 dm3kg−1 12–20 g/L H2 = 58.7% CO = 10.6%

References [20] [21] [22] [23] [24]

2.2. Second Generation (2G) Figure 5 provides the 2G biofuels feedstock. Second-generation biofuels address

many of the issues related to first-generation biofuels. The prospects for fostering regional growth and improving the economic situation in developing regions is envisaged with 2G biofuels. Around the world, various strategies for the production of second-generation biofuels are being considered. Still, the focus is primarily on two distinct paths, either the thermo or bio route generated by biomass of cellulose and lignin, tree surplus, and sea-sonal forage crop. Thermochemical manufacturing has the significant benefit of higher versatility of feedstock than biological production. The “thermo” route is focused on the heat processing of biomass under decreased oxidizing agent concentrations. Under the temperature range of 300 to 1000 °C, the bottom range will primarily produce solid biofuel called biochar. At elevated temperatures, pyrolytic oil and syngas are the most concen-trated substances in the middle range. The “bio” approach includes the pretreatment of lignocellulosic material, enzymatic hydrolysis, and the fermentation of sugars by specific strains of microorganism. It is more challenging to convert lignocellulose to reducing sug-ars than it is to convert starch. Biological, physical (thermal), or chemical catalysts are used to pretreat biomass in the biochemical pathway as shown in Figure 6; hence, enhanced advances in the development of 2G biofuels are hampered due to the chemical and struc-tural properties of the extracellular matrix. The biodiesel yield parameters from the 2G biofuel are highlighted in Table 2.

Figure 5. Feedstock of second-generation biofuels.

Second generation

Bioethanol from crops

Lignocellulosic Biomass

Biodiesel from Fat

Waste Vegetable Oil

Figure 5. Feedstock of second-generation biofuels.

Table 2. Parameter and yield of biodiesel, bioethanol, biomethane, and syngas from various feedstocks of second-generation biofuels.


Feedstock Palm oil Sugarcane bagasse Corn Stover Rice straw Corn Stover

ParametersTransesterification,H2SO4—5% v/w,

95 ◦C/540 min

Fermentation, Acid(H2SO4)

hydrolysis,Kluyveromyces sp.

IIPE453, Fermentationat 50 ◦C

Anaerobicdigestion,Cellulase

(Spezyme CP)T = 37 ± 1 ◦C,

t = 30 days

Fermentation, C.sporogenes BE01,

(37◦ C and 6.7 pH)

Gasification,Fluidized bed gasifier

GA: steam;T: 600–710 ◦C;

ER: N.A.

Yield 97 w/w 165 g/kg 135 dm3 kg−1 VS 5.52 g/LH2: 26.9,CO: 24.7

CO2: 23.7 CH4: 15.3

References [25] [26] [27] [28] [29]

New technologies have focused on genomes as well as structural and artificial ge-netics that would offer demanding opportunities for enhancing the digestibility of cellwalls [30] and have the potential to raise PFCE from biomass radically. In species suchas Caldicellulosiruptor saccharolyticus and Acidothermus cellulolyticus [31], enzymes haverecently been used to degrade lignocellulose and speed up the process. The efficiencyof cellulosic biofuels can also be significantly strengthened by supplying engineered mi-crobes with the potential to digest lignocellulosic biomass even without the application ofcostly enzymes. Some see genetic modification (GM) techniques as key to achieving highyield, thus boosting the total energy stability of crop residues, for example, developingresistance to fertilizers and pesticides and flooding. However, GM discussions may resultfrom socioeconomic and political decisions debates [32]. Second-generation biofuels arecontemporary and innovative, but they do have a specific impact on sustainability. Thebalance of the life cycle of GHG emissions remains a problem depending on the locationof the 2G biofuels produced, the conservation methods, the modes of transportation, andthe methods of processing. The second-generation biofuels include waste from operations,


such as methane from garbage dumps or the conversion of waste from processes derivedfrom fossil fuels [33]. A detailed study by Havlík et al. [34] (reported that 2G biofuel pro-duction powered by wood from clean sources would reduce overall emissions, consideringthe deforestation, agriculture water consumption, and increased crop prices especiallywith the rise of biofuel land area. Still, some other environmental requirements, such asecosystem preservation, climate regulation, and fuelwood availability to the inhabitants,could be influenced by biomass feedstocks and land use [34].


Figure 6. Pretreatment of lignocellulosic material.

Table 2. Parameter and yield of biodiesel, bioethanol, biomethane, and syngas from various feedstocks of second-genera-tion biofuels.

Biofuels Biodiesel Bioethanol Biomethane Biobutanol Syngas Feedstock Palm oil Sugarcane bagasse Corn Stover Rice straw Corn Stover

Parameters Transesterification,

H2SO4—5%v/w, 95 °C/540 min

Fermentation, Acid (H2SO4)

hydrolysis, Kluyveromycessp.

IIPE453, Fermentation at 50°C

Anaerobic digestion, Cellulase

(Spezyme CP) T = 37 ± 1 °C, t = 30 days

Fermentation, C. sporogenes BE01,

(37° C and 6.7 pH)

Gasification, Fluidized bed gas-ifier GA: steam; T:

600–710 °C; ER: N.A.

Yield 97 w/w 165 g/kg 135 dm3 kg–1 VS 5.52 g/L

H2: 26.9, CO: 24.7

CO2: 23.7 CH4: 15.3

References [25] [26] [27] [28] [29]

New technologies have focused on genomes as well as structural and artificial genet-ics that would offer demanding opportunities for enhancing the digestibility of cell walls [30] and have the potential to raise PFCE from biomass radically. In species such as Cald-icellulosiruptor saccharolyticus and Acidothermus cellulolyticus [31], enzymes have recently been used to degrade lignocellulose and speed up the process. The efficiency of cellulosic biofuels can also be significantly strengthened by supplying engineered microbes with the potential to digest lignocellulosic biomass even without the application of costly enzymes. Some see genetic modification (GM) techniques as key to achieving high yield, thus boost-ing the total energy stability of crop residues, for example, developing resistance to ferti-lizers and pesticides and flooding. However, GM discussions may result from socioeco-nomic and political decisions debates [32]. Second-generation biofuels are contemporary and innovative, but they do have a specific impact on sustainability. The balance of the life cycle of GHG emissions remains a problem depending on the location of the 2G bio-fuels produced, the conservation methods, the modes of transportation, and the methods of processing. The second-generation biofuels include waste from operations, such as me-thane from garbage dumps or the conversion of waste from processes derived from fossil fuels [33]. A detailed study by Havlík et al. [34] (reported that 2G biofuel production pow-ered by wood from clean sources would reduce overall emissions, considering the defor-estation, agriculture water consumption, and increased crop prices especially with the rise of biofuel land area. Still, some other environmental requirements, such as ecosystem

Figure 6. Pretreatment of lignocellulosic material.

The bacteriological and physical properties of soils are also adversely affected by theremoval of crop and forestry residues. For instance, according to Powlson et al. [35] theenergy savings related to burying wheat bran in croplands are greater to those produceddue to their removal for biodiesel production [35]. Various published data indicate thatwhen forest deadwood is removed, the occurrence and variability of bird species decreasesignificantly. For cavity nesters (e.g., woodpeckers), the critical impact was recorded(e.g., woodpeckers). The eggs and offspring of insects are captured and expelled fromthe forest as waste is removed and shipped to energy plants, not only reducing insectproliferation but also removing an important source of bird food [36,37]. Evidently, overthe past decade(s), the growth of cellulosic biofuel plants on an industrial level has becomeweaker than anticipated. The latest biofuel output cost statistics indicate that biofuels ofthe second generation on an energy-average basis are two to three times more expensivethan fossil fuels [10].

Currently, producing 2G biofuel is cost-efficient, but there seem to be various tech-nological challenges that need to be overcome before realizing their potential. Duringthe pretreatment and extraction phase, the need for thermal energy and enzymes duringcellulose-based hydrolysis increases the production value of bioethanol [38]. The treatmentof co-products such as wheat bran raises significant sustainability concerns because pre-treatment for enzymatic saccharification is required to overcome the issues associated withlignocellulosic biomass [39]. In addition to the production of sustainable, low pretreatmentmethods and highly productive fermentation processes, the incorporation of hemicelluloseintegration is a technical factor that should be taken into account when developing sus-tainable biorefineries [40]. Worldwide, several efforts are underway to commercialize thesecond-generation biofuels provided from both routes’ extraction. The second-generationmainly operates on a pilot or prototype scale, as shown in Table 3.


Table 3. Commercialization status of advanced biofuel production route [41].

Conversion Process Pilot/Demonstration Demonstration Small

Commercial Commercial

HEFA X

Gasification—FT X

Pyrolysis and upgrading X

HTL and upgrading X

Advanced sugar fermentation to hydrocarbons X

Ethanol production from agricultural residues(pretreatment, enzymatic hydrolysis, and fermentation) X

In 2007 and 2012, the Canadian company Iogen corporation operated a demonstrationplant and then planned to develop a production plant in Brazil that could manufactureethanol in 40 million liters of production by sugar cane bagasse [42]. Even so, since theirtechnology was not advanced enough or failed in their start-up process, several other firmshad to close down [43,44]. When compared to the fossil energy, they could replace the basiscost of production. However, they are still just too expensive to manufacture. Changesin policy could expedite the transition from first-generation biofuels to the commercialdeployment and adoption of second-generation biofuels. However, regulations must bedesigned to encourage the development of the most favorable biofuels while discouragingthe production of “poor” biofuels [8]. Table 4 highlights the difference between first-generation and second-generation biofuels.

Table 4. First-generation vs. second-generation biofuels.

Conversion Process FirstGeneration

SecondGeneration

Possibilities for greenhouse gases mitigation - X

Ability to reduced consumption of fossil fuels - X

The viability of using marginal land to produce feedstock - X

High manufacturing value X -

Relatively simple conversion procedure X -

High prospects for a net decrease in the use of non-renewable resources X -

More output of land use - X

2.3. Third-Generation Biofuel (3G)

First-generation and second-generation biofuels are not exclusively biological norreliant on environmentally sustainable feedstocks. Furthermore, high-energy inputs arerequired in both feedstock processing and biofuel synthesis. Despite the popularity of1G biofuels, they suffer from limitations due to disrupting the chain of food and feed,while second-generation feedstocks are losing reputation owing to the increased cost ofthe synthesis and chemical processing of biodiesel. The production of greener and moreefficient biofuels is essential to meet the challenge of entirely replacing conventional fossilfuels with third-generation biofuels. The biofuel yield from 3G feedstock is highlighted inTable 5. Figure 7 provides the third generation feedstock.



Table 4. First-generation vs. second-generation biofuels.

Conversion Process First Generation

Second Generation

Possibilities for greenhouse gases mitigation - Ability to reduced consumption of fossil fuels -

The viability of using marginal land to produce feedstock - High manufacturing value -

Relatively simple conversion procedure - High prospects for a net decrease in the use of non-renewable

resources -

More output of land use -

2.3. Third-Generation Biofuel (3G) First-generation and second-generation biofuels are not exclusively biological nor re-

liant on environmentally sustainable feedstocks. Furthermore, high-energy inputs are re-quired in both feedstock processing and biofuel synthesis. Despite the popularity of 1G biofuels, they suffer from limitations due to disrupting the chain of food and feed, while second-generation feedstocks are losing reputation owing to the increased cost of the syn-thesis and chemical processing of biodiesel. The production of greener and more efficient biofuels is essential to meet the challenge of entirely replacing conventional fossil fuels with third-generation biofuels. The biofuel yield from 3G feedstock is highlighted in Table 5. Figure 7 provides the third generation feedstock.

Figure 7. Third-generation biofuels.

Table 5. Parameters and yield of biodiesel, biomethane, bioethanol, and syngas synthesis from various feedstocks of third-generation biofuels.


Feedstock Spirulina platensis Chlorococcum infu-sionum

S. latissima (macroal-gae) Macroalgae Spirulina

Parameters

Transesterification, reac-tion temperature 55 °C, 60% catalyst concentra-

tion, 1:4 algae biomass to methanol ratio, 450 rpm

stirring intensity

Fermentation, alka-line pretreatment,

temp. 120 °C, S. cerevisiae

Anaerobic digestion, 53 °C for a period of 34

days, flushed with N2/CO2 (80/20%), to

obtain anaerobic condi-tions.

Fermentation, C. beijerinckii ATCC

35702, (37 °C and 6.0 pH).

Pyrolsis, Temp = 550 °C.

Yield 60 g/kg 260 g /kg 340 ± 48.0 mL g VS −1 4 g/L

H2 = 29, CO = 24, CO2 = 24, CH4 = 18

References [45] [26] [46] [47] [48]

Algae are at the forefront of the production of third-generation biofuel. When algae are used to produce biofuels, CO2 emissions will be reduced in comparison to sources of

Third generation

Microalgae Macroalgae

Figure 7. Third-generation biofuels.

Table 5. Parameters and yield of biodiesel, biomethane, bioethanol, and syngas synthesis from various feedstocks ofthird-generation biofuels.


Feedstock Spirulina platensis Chlorococcuminfusionum S. latissima (macroalgae) Macroalgae Spirulina

Parameters

Transesterification, reactiontemperature 55 ◦C, 60%

catalyst concentration, 1:4algae biomass to methanol

ratio, 450 rpm stirringintensity

Fermentation,alkaline pretreatment,

temp. 120 ◦C,S. cerevisiae

Anaerobic digestion,53 ◦C for a period of34 days, flushed withN2/CO2 (80/20%), to

obtain anaerobicconditions.

Fermentation,C. beijerinckiiATCC 35702,

(37 ◦C and 6.0 pH).

Pyrolsis,Temp = 550 ◦C.

Yield 60 g/kg 260 g /kg 340 ± 48.0 mL g VS −1 4 g/L

H2 = 29,CO = 24,CO2 = 24,CH4 = 18

References [45] [26] [46] [47] [48]

Algae are at the forefront of the production of third-generation biofuel. When algaeare used to produce biofuels, CO2 emissions will be reduced in comparison to sources offossil fuels, reducing rising temperatures. The production of 1 kg of microalgae, accordingto Chisti [49], involves the fixation of up to 1.8 kg CO2. This unique necessity for biomassproduction has evolved microalgae to focus on intensive bio-mitigation studies, whichmay vastly enhance life-cycle savings. The manufacture of biofuels through algae highlydepends upon lipid value. Fast-growing algae are thought to have low oil content, whileslow-growing algae have high lipid content [50]. Therefore, microalgal strains selectionwith greater efficiencies and a rapid growth rate of metabolites is essential [51]. Greenmicroalgae (Chlorophyta) collect oil at a higher rate than other algal taxa such as cyanobac-teria, brown algae, and red algae [52]. Owing to high lipids, approximately 60–70% [53]and high efficiency, 7.4 g/L/day for Chlorella protothecoides [54], species such as Chlorellaare evidently targeted. Biofuel production through algal biomass could be commerciallyviable when algal products and waste are flexibly used. A variety of methods can be usedto transform microalgae biomass into energy sources (Figure 8).

LCA analysis was conducted on the use of macroalgae for increased CO2 fixation andbiofuel generation [55]. It was showed that increased CO2 fixation by macroalgae couldprovide an energy advantage linked with carbon recycling [55]. In the best-case scenariothus far studied, macroalgae can yield a net energy of 11,000 MJ/t dry algae compared to9500 MJ/t for microalgae gasification. Lam and Lee [56] evaluated the energy-efficiencyratio (EER) of agricultural and microalgae-based biofuel manufacturing techniques. TheEER is defined as the energy output divided by the energy intake. EERs for crop-basedbiofuel ranged from 1.44 to 5, whereas EERs for microalgae-based biofuel ranged from0.35 to 434 [56]. Overall, the EER for microalgae-based biofuel generation was lower,but this ratio may rise if the process continues to develop. Microalgae-based biodieselfuels have density, viscosity, flash point, heating value, cold filter clogging point, andsolidifying point in common with petroleum-based biofuels. As a result, they meet boththe American Society for Testing and Materials (ASTM) and the International BiodieselStandard for Vehicles (IBSV) requirements [57]. Table 6 compares the properties of macroand micro-algae based biofuels.



fossil fuels, reducing rising temperatures. The production of 1 kg of microalgae, according to Chisti [49], involves the fixation of up to 1.8 kg CO2. This unique necessity for biomass production has evolved microalgae to focus on intensive bio-mitigation studies, which may vastly enhance life-cycle savings. The manufacture of biofuels through algae highly depends upon lipid value. Fast-growing algae are thought to have low oil content, while slow-growing algae have high lipid content [50]. Therefore, microalgal strains selection with greater efficiencies and a rapid growth rate of metabolites is essential [51]. Green microalgae (Chlorophyta) collect oil at a higher rate than other algal taxa such as cyano-bacteria, brown algae, and red algae [52]. Owing to high lipids, approximately 60–70% [53] and high efficiency, 7.4 g/L/day for Chlorella protothecoides [54], species such as Chlo-rella are evidently targeted. Biofuel production through algal biomass could be commer-cially viable when algal products and waste are flexibly used. A variety of methods can be used to transform microalgae biomass into energy sources (Figure 8).

Figure 8. Biofuel generation from microalgae.

LCA analysis was conducted on the use of macroalgae for increased CO2 fixation and biofuel generation [55]. It was showed that increased CO2 fixation by macroalgae could provide an energy advantage linked with carbon recycling [55]. In the best-case scenario thus far studied, macroalgae can yield a net energy of 11,000 MJ/t dry algae compared to 9500 MJ/ t for microalgae gasification. Lam and Lee[56] evaluated the energy-efficiency ratio (EER) of agricultural and microalgae-based biofuel manufacturing techniques. The EER is defined as the energy output divided by the energy intake. EERs for crop-based biofuel ranged from 1.44 to 5, whereas EERs for microalgae-based biofuel ranged from 0.35 to 434. [56]. Overall, the EER for microalgae-based biofuel generation was lower, but this ratio may rise if the process continues to develop. Microalgae-based biodiesel fuels have density, viscosity, flash point, heating value, cold filter clogging point, and solidify-ing point in common with petroleum-based biofuels. As a result, they meet both the American Society for Testing and Materials (ASTM) and the International Biodiesel Stand-ard for Vehicles (IBSV) requirements [57]. Table 6 compares the properties of macro and micro-algae based biofuels .

Figure 8. Biofuel generation from microalgae.

Table 6. Properties of micro and macro-algal biodiesel as compared to conventional diesel [58,59].

Fuel Property Unit MicroalgaeBiodiesel

MacroalgaeBiodiesel

Biodiesel StandardEN 14214 Diesel

Cetane Number - 46.5 58.23 51 53.3

Kinematic Viscosity @40 ◦C mm 2/s 5.06 4.3 3.5–5.0 2.64

Density @15 ◦C kg/L 0.912 0.868 0.86–0.90 0.84

Acid Value mg KOH/g 0.14 0.13 0.5 max 0

Flashpoint ◦C - 155 101 min 100 1-D126 2-D

Cloud Point ◦C 16.1 −4 - 4

Sulfur Content mg/kg 7.5 8.9 10 max 5.9

Copper Strip Corrosion(3 h at 50 ◦C) - 1 1 1 1

Approximately 30% is the algal biomass’s oil portion, and the leftover 70% is the algaeby-product. This by-product can be used for medical chemicals, cosmetics, toiletries, andfragrance products.

High energy and cost-intensive downstream processes, such as enzymatic hydrolysisand metabolic pathway extraction, remain primary techno-economic obstacles to the fullcommercialization of microalgal biodiesel production [60]. Efroymson et al. [61] suggestedthat by reducing the number of phases in the manufacturing and co-production of amore energetic fraction, the value of algal biofuels could be dramatically lowered. Manyalgae specimens are not appropriate for industrial cultures, as the structure of microalgallipids in fatty acids may not be ideal when used as biofuel. In challenging situations, theaccumulation of lipids leads to cell development and division being stopped, resulting in aclear limitation of the productivity of biomass [62]. Genetic manipulation engineering candeliver innovative routes to lipid and algal biomass production [63]. Through calculation,it was demonstrated that replacing 1% of US road fuel source with macroalgal biofuel onlyinvolves 0.09% area of the Exclusive Economic Zone (EEZ) [64]. Such a prospect is mostlikely to remain on a document before promoting strategies are carried out. Microalgae-derived jet fuels have also been extensively tested in commercial and military aircraft.Solazyme Inc. produced the world’s first jet fuel made entirely of algae using the UOPHEFA process technology and fermentation. The US Navy has tested Solazyme’s jetfuel [64]. To analyze the environmental effect of an algal-based BAF supply chain in the


United States, Agusdinata and Laurentis [65] combined LCA and multi-actors (stakeholderdecisions) found that algal biofuels have the potential to reduce the country’s aircraftindustry’s life-cycle CO2 emissions by up to 85% by 2050 [65]. Massive algae processingalso faces technical and logistical challenges, but respondents believe that algae-basedbiofuels can play an important role in the advancement.

Emerging Trend of Nanotechnology

Nanotechnology, as a creative and ground-breaking technique, has a broad arrayof applications and prominent roles. NPs are reliable, cost-effective, and environmen-tally sustainable, with high stability, a faster synthesis rate, and a simple procedure. Ananoparticle is described as a structure with a diameter of 0.1 to 100 nm. As a result oftheir extraordinary physicochemical properties, nanoparticles are now being used strategi-cally in biofuel development. Many nanomaterials with unique properties such as TiO2,Fe3O4, SnO2, ZnO, sulfur, graphene, and fullerene have been used in biofuel processing.Nanoparticle-aided microalgal harvesting has become the new trend to enhance energyusage, total microalgal concentration, quality, and process cost [66]. In a large-scale sam-ple, the use of nanoparticles on microalgae harvesting claimed a 20–30% reduction inmicroalgae production cost [67]. The use of nanoparticles in bio-diesel blended fuels hasimproved efficiency and lowered emissions. More emphasis should be put on this inthe future. Karthikeyan et al. have concentrated on preparing various biodiesel blendsusing CeO2 additives in the hope of long-term applications in single-cylinder compressionignition engines that would benefit the whole population [68]. In the presence of ion–silicananocomposites, algal oils have a high yield of production [69]. The Ames Laboratoryhas created a new technique dubbed “nano-farming” that extracts oil from algae usingsponge-like mesoporous nanoparticles. The process does not damage the algae in the sameway that other methods are being produced, lowering production costs and shortening theproduction cycle [70]. Table 7 highlights the use of nanoparticles in algal biofuels.

Table 7. Role of various nanoparticles in algal biofuels.

Nanoparticles Functions Ref.

Al2O3.Improved the Chlorella sp. microalgae growth by 18.9% after 4 days of exposure to a

concentration of 1000 mg L−1 Al2O3[71]

TiO2Improved total yield in microalgae processing (e.g., cell

suspension, cell division, and cell harvesting) [67]

Fe3O4Increased harvesting productivity by 95% of Scenedesmus

ovalternus and Chlorella vulgaris when grown with iron oxide NP [72]

CaO During scaled-up catalytic transesterification, the conversionefficiency of biodiesel was 91% [73]

TiO2, CeO2 Enhance 10–11% of biogas yield from wastewater treatment [74]

Although nano-additive applications were essential to microalgae growth, harvesting,biofuel conversion, and biofuel applications to enhance efficiency, there were still certainobstacles prior to the deployment of nano-additives for the commercialization scale. Nano-additives in micro-algal biofuels are limited to the laboratory and pilot size, which is a sig-nificant constraint. Despite its benefits, one of the primary difficulties with NPs is the highcost of manufacture, which has hampered the commercialization of nanofluids (Figure 9).



SnO2, ZnO, sulfur, graphene, and fullerene have been used in biofuel processing. Nano-particle-aided microalgal harvesting has become the new trend to enhance energy usage, total microalgal concentration, quality, and process cost [66]. In a large-scale sample, the use of nanoparticles on microalgae harvesting claimed a 20–30% reduction in microalgae production cost [67]. The use of nanoparticles in bio-diesel blended fuels has improved efficiency and lowered emissions. More emphasis should be put on this in the future. Karthikeyan et al. have concentrated on preparing various biodiesel blends using CeO2 additives in the hope of long-term applications in single-cylinder compression ignition engines that would benefit the whole population [68]. In the presence of ion–silica nano-composites, algal oils have a high yield of production [69]. The Ames Laboratory has cre-ated a new technique dubbed “nano-farming” that extracts oil from algae using sponge-like mesoporous nanoparticles. The process does not damage the algae in the same way that other methods are being produced, lowering production costs and shortening the production cycle [70]. Table 7 highlights the use of nanoparticles in algal biofuels

Table 7. Role of various nanoparticles in algal biofuels.

Nanoparticles Functions Ref.

Al2O3. Improved the Chlorella sp. microalgae growth by 18.9% after 4 days of exposure to a concentration of 1000 mg L−1 Al2O3 [71]

TiO2 Improved total yield in microalgae processing (e.g., cell suspension, cell division, and cell harvesting) [67]

Fe3O4 Increased harvesting productivity by 95% of Scenedesmus ovalternus and Chlorella vulgaris when grown with iron oxide NP [72]

CaO During scaled-up catalytic transesterification, the conversion efficiency of biodiesel was 91% [73]

TiO2, CeO2 Enhance 10–11% of biogas yield from wastewater treatment [74]

Although nano-additive applications were essential to microalgae growth, harvest-ing, biofuel conversion, and biofuel applications to enhance efficiency, there were still cer-tain obstacles prior to the deployment of nano-additives for the commercialization scale. Nano-additives in micro-algal biofuels are limited to the laboratory and pilot size, which is a significant constraint. Despite its benefits, one of the primary difficulties with NPs is the high cost of manufacture, which has hampered the commercialization of nanofluids (Figure 9).

Figure 9. Application of nanoparticles. Figure 9. Application of nanoparticles.

2.4. Fourth-Generation Biofuel (4G)

The most promising advanced biofuels are those from the fourth generation of biofuels.The feedstocks of the fourth-generation biofuels are genetically engineered microalgae,microbes, yeast, and cyanobacteria; these microorganisms are genetically engineered. Thebest way to cut the price, nutrients consumption, and ecological footprint is to boost produc-tivity and lipid accumulation. Ketzer et al. found that from a biological standpoint, a betterenergy return on investment (EROI) might be obtained by improving photo-conversionefficiency, which would result in higher biomass and energy yields. Increasing and al-tering the buildup or release of energy products (e.g., lipids, alcohol) is currently beingresearched [75]. Genome editing strategies are frequently used to improve the efficiencyand lipid composition of algae. Currently, three types of genetic modification tools arecommonly used for genomic editing of microalgae strains: zinc-finger nuclease (ZFN), tran-scription activator-like effector nucleases (TALEN), and clustered frequently interspacedpalindromic sequences (CRISPR/Cas9) [76]. As a result of the complexity and difficultyof the experimental design of ZFN and TALEN, the CRISPR-Cas9 method is the most ac-tively developed in microalga [77,78]. Wang et al. performed precise CRISPR/Cas9-basedgenome editing of commercial algal strains such as Nannochloropsis, which accumulates oilas a source of plant-like fats for biofuel generation under nitrogen shortage [79]. EngineeredZFNs were utilized by Sizova et al. [80]) and Greiner et al. [81] to target the COP3 and COP4genes in C. reinhardtii. The effectiveness of the ZFNs was only observed in the tailoredmodel strain of C. reinhardtii. The most difficult challenge is to generate unique ZFNswith high specificity and affinity for the target sites [80,81]. Before executing the actualexperiment, ZFNs must be validated using a gene-targeting selection method [80]. Usinggenetic and metabolic engineering, it is possible to connect the third and fourth generationscompared to 3G biofuels where the main focus is on the production of biomass of algaeto generate biodiesel. On the other side, the most attractive feature of fourth-generationbiofuel is introducing the incorporation of modified photosynthetic microorganisms [82].Figure 10 shows the process of biofuel production from genetically modified algae.



2.4. Fourth-Generation Biofuel (4G) The most promising advanced biofuels are those from the fourth generation of bio-

fuels. The feedstocks of the fourth-generation biofuels are genetically engineered micro-algae, microbes, yeast, and cyanobacteria; these microorganisms are genetically engi-neered. The best way to cut the price, nutrients consumption, and ecological footprint is to boost productivity and lipid accumulation. Ketzer et al. found that from a biological standpoint, a better energy return on investment (EROI) might be obtained by improving photo-conversion efficiency, which would result in higher biomass and energy yields. In-creasing and altering the buildup or release of energy products (e.g., lipids, alcohol) is currently being researched [75]. Genome editing strategies are frequently used to improve the efficiency and lipid composition of algae. Currently, three types of genetic modifica-tion tools are commonly used for genomic editing of microalgae strains: zinc-finger nu-clease (ZFN), transcription activator-like effector nucleases (TALEN), and clustered fre-quently interspaced palindromic sequences (CRISPR/Cas9) [76]. As a result of the com-plexity and difficulty of the experimental design of ZFN and TALEN, the CRISPR-Cas9 method is the most actively developed in microalga [77,78]. Wang et al. performed precise CRISPR/Cas9-based genome editing of commercial algal strains such as Nannochloropsis, which accumulates oil as a source of plant-like fats for biofuel generation under nitrogen shortage [79]. Engineered ZFNs were utilized by Sizova et al. [80]) and Greiner et al. [81] to target the COP3 and COP4 genes in C. reinhardtii. The effectiveness of the ZFNs was only observed in the tailored model strain of C. reinhardtii. The most difficult challenge is to generate unique ZFNs with high specificity and affinity for the target sites [80,81]. Be-fore executing the actual experiment, ZFNs must be validated using a gene-targeting se-lection method [80]. Using genetic and metabolic engineering, it is possible to connect the third and fourth generations compared to 3G biofuels where the main focus is on the pro-duction of biomass of algae to generate biodiesel. On the other side, the most attractive feature of fourth-generation biofuel is introducing the incorporation of modified photo-synthetic microorganisms [82]. Figure 10 shows the process of biofuel production from genetically modified algae.

Figure 10. Fourth-generation biofuel production.

2.4.1. Microorganisms Use in Fourth-Generation Biofuel

In biofuel processing, several microbes, yeast, cyanobacteria, and microalgae are used,with cyanobacteria and microalgae being the best choices for this reason. The selectionof suitable strains, as all microbial species, cannot be genetically modified due to thecomplexity of structure, high nutrient demand, or environmental intolerance. Tables 8–10provides the lipid yield, risk-mitigation and yield parameters of the GM algae

Green Algae

The Chlamydomonas reinhardtii has been genetically engineered to express many es-sential biofuel characteristics [83]. However, the production rate of biomass is low. Someexamples of green algal such as Chlorella, Parachlorella, Nannochloropsis, Scenedesmus, Botry-ococcus, and Neo-chloris are rich in lipid content and hence mostly use for biofuel instead ofhaving low biomass.

Blue-Green Algae

Cyanobacteria are among the first microorganisms to have lasted for a few billionyears. They are a crucial source of atmospheric oxygen and play a vital role in the dailylives of ordinary people [84]. There are many possible uses for cyanobacteria, such asfeed sources, agricultural biofertilizers, and wastewater treatment [85]. Compared to otherphotoautotrophs, biofuel production from cyanobacteria has a lot of potential as a biofuelplatform, since they do not need fermentable sugar or arable land to grow. They willhave far less competitiveness with farmland capacity to fix carbon dioxide gas. Genetictractability, horizontal gene transfer, and competitiveness among genetically modifiedcyanobacteria and other microorganisms may impact natural ecosystems [61,86]. Syne-chocystis sp. PCC 6803, Synechococcus elongatus sp. PCC 7492, Synechococcus sp. PCC 7002,and Anabaena sp. PCC 7120 all have been used as model organisms for genetic engineering.The optimal production host, on the other hand, is challenging to forecast [87]. Fourth-generation processes include pyrolysis (at temperatures ranging from 400 to 600 ◦C [88]),gasification, and solar-to-fuel pathways in addition to genetic modification [89].


Table 8. Genetically engineered microalgae generate lipids for biofuel production.

Species Attainments Reference(s)

Chlamydomonas reinhardtii Increased lipid content by 1.5 times20% increase in triglyceride content [83,90]

Phaeodactylum tricornutum 35% increase in lipid1.1 times increase in triglyceride content [91,92]

According to Snow and Smith [93], genetically engineered microalgae-induced compli-cations include future environmental challenges such as ecological changes, toxicity, lateraltransference of genes, and competition with native organisms [94]. The disposal of GMOsis one of the critical issues with their use. Since the intentional or unintentional release ofchromosomal or plasmid DNA at specific concentrations might result in horizontal genetransfer through transformation, there are stringent rules for its disposal [95].

Table 9. Risk mitigation for GM algae.

Biological Method Physical Method

Inactivation of a gene that regulates sexual behavior To assess possible or unknown hazards, controlled field experimentshould be conducted.

Upon escape, terminator gene expressed UV, chemical, and heat deactivation during the harvesting ofgenetically modified algae.

To reduce the danger of large-scale GM algae being released into the environment, twomain containment strategies are being considered: first, physically stopping the algae fromescaping into the atmosphere, and second, genetically preventing the algae from replicatingand competing in nature [96]. GE algae outperform native strains in the context of ecologicalcompatibility and cost-effectiveness, making algal biofuels more viable. There is proof oftheir superiority and the absence of significant drawbacks on a lab and prototype size, butthis must be demonstrated commercially, since it is necessary for the genetic stability ofGE algae. Although CRISPR technology eliminates the fear of GMOs, it is not universallyembraced. For example, gene-edited organisms must be subjected to the same onerousrestrictions as traditional GMOs, according to a judgment by the European Union’s Courtof Justice ECJ [97].

Table 10. Parameters and yield of biodiesel, biomethane, bioethanol, and syngas synthesis from various feedstocks offourth-generation biofuels.


Feedstock E. coli Synechocystis sp. PCC6803 - E. coli -

Parameters

Deletion of Aas gene in strain SS3B to producestrain SS34.

Introduced DmJHAMT(Drosophila melanogasterJuvenile Hormone Acid

O-Methyltransferase)inlet temperature 250 ◦C with split ratio 1:1;carrier gas: helium; flow: 5 mL/min; oventemperature: initial temperature of 160 ◦C,hold 3 min; gradient to 255 ◦C at 5 ◦C/min;

hold 3 min; inlet temp: 270 ◦C, detectortemp: 330 ◦C

Integration of pyruvatedecarboxylase from Z.

mobilis and endogenousalcohol dehydrogenaseslr1192 under control of

different promoters.Temperature: 37 ◦C,shaking at 80 rpm,induced by 0.4 mM

isopropylthiogalactoside

-

Deletion of adh, ldh, frd,fnr and pta and insertion

of bcd-etfAB from C.acetobutylicum.

Cultures grownsemi-aerobically in shake

flasks at 37 ◦C for 24 h

-

Yield 0.56 g/L 5.50 g/L 0.37 g/L

References [98] [99] [100]


Industries Involved in Third and Fourth Generation Biofuels

The Scottish Association for Marine Science (SAMS) is working on MacroFuels, aHorizon 2020 project to develop improved biofuels from seaweed or macroalgae. Theproject’s goal is to make a breakthrough in biofuel production. It also aims to developtechnologies for fuels that can be utilized in heavy transportation and aircraft.

Craig Ventor’s Synthetic Genomics is currently developing microbes capable of pro-ducing fuel directly from carbon dioxide (CO2). By 2050, it is predicted that the fourthgeneration will be wholly developed and play a significant role in the global power industry.A brief comparison of 3G and 4G biofuels are presented in Table 11.

Table 11. Comparisons between third and fourth-generation biofuels.

Biofuel Generation Third Generation Fourth Generation

Biomass used Algae and microorganism Engineered crops and solar fuels

Processing methodologyBiochemical conversion,

Chemical reaction, Directcombustion, Thermochemical conversion

Genetically modified algae,Biochemical conversion,

Thermochemical conversion

Generated fuel Methane, Bioethanol, Biobutanol,Syngas, Biodiesel

Methane, Bioethanol, Biobutanol,Biodiesel, Syngas

Advantages Easy to cultivateNo competition for food crops

Biomass and production yield both are high.Increase CO2 absorption capacity

3. Sustainable Assessment of Third-Generation Biofuels

The sustainability concept is multidimensional. It acknowledges that there are in-herent relations between economic, social, and environmental well-being as shown inFigure 11. If any one of the dimensions changes, it will have an impact upon the othertwo dimensions. Sustainable biofuel production should include preserving biodiversity,sustainable water utilization, healthy air quality, soil conservation, social issues (such asstorage, transportation, health effects, etc.), and most importantly, fair labor practices. Onthe one hand, biofuel contributes to the prospects of CO2 reduction, improves air quality,and provides net energy gain.


Chemical reaction, Direct combustion, Thermochemical

conversion

Generated fuel Methane, Bioethanol, Biobutanol,

Syngas, Biodiesel Methane, Bioethanol, Biobutanol,

Biodiesel, Syngas

Advantages Easy to cultivate No competition for food crops

Biomass and production yield both are high.

Increase CO2 absorption capacity

3. Sustainable Assessment of Third-Generation Biofuels The sustainability concept is multidimensional. It acknowledges that there are inher-

ent relations between economic, social, and environmental well-being as shown in Figure 11. If any one of the dimensions changes, it will have an impact upon the other two di-mensions. Sustainable biofuel production should include preserving biodiversity, sustain-able water utilization, healthy air quality, soil conservation, social issues (such as storage, transportation, health effects, etc.), and most importantly, fair labor practices. On the one hand, biofuel contributes to the prospects of CO2 reduction, improves air quality, and pro-vides net energy gain.

On the other hand, the continuous production of biofuels harms biodiversity, causes soil degradation, and affects food security. Since biodiesel production has risen steadily globally, food prices for vegetable oils have increased significantly [101]. Studies on ma-rine algal biofuels have received interest in the last few decades. A potential solution to energy and environmental problems is a commercially feasible algal cultivation. It is cost-effective, requires no additional land, uses less water, and reduces atmospheric CO2. The global efficiency, net productivity per hectare, avoided CO2 emissions, net present value (NPV), and levelized cost of energy (LCOE) are the key metrics used for the sustainability evaluation of biofuels. Third-generation biofuels, with higher pollution reductions, aim to be more sustainable. These biofuels are focused on biomass sources that are not used for other primary purposes, such as food processing and cultivation. Algae demonstrate great promise as a possible future green energy source because of their environmental friendli-ness and high oil-yielding ability per given field.

Figure 11. Sustainability aspects of biofuel production.

Sustainability

Social

Employability

Land Issues

Small holder integration

Food Security

Environmental

GHG Emission reduction

Soil Quality

Water use and quality

Biodiversity

Economic

Energy Sufficiency and self sufficiency

Balance of payments

Financing

Fuel Cost

Figure 11. Sustainability aspects of biofuel production.

On the other hand, the continuous production of biofuels harms biodiversity, causessoil degradation, and affects food security. Since biodiesel production has risen steadilyglobally, food prices for vegetable oils have increased significantly [101]. Studies onmarine algal biofuels have received interest in the last few decades. A potential solution toenergy and environmental problems is a commercially feasible algal cultivation. It is cost-


effective, requires no additional land, uses less water, and reduces atmospheric CO2. Theglobal efficiency, net productivity per hectare, avoided CO2 emissions, net present value(NPV), and levelized cost of energy (LCOE) are the key metrics used for the sustainabilityevaluation of biofuels. Third-generation biofuels, with higher pollution reductions, aimto be more sustainable. These biofuels are focused on biomass sources that are not usedfor other primary purposes, such as food processing and cultivation. Algae demonstrategreat promise as a possible future green energy source because of their environmentalfriendliness and high oil-yielding ability per given field.

3.1. Land

The primary goal of biofuel conservation is to conserve land. Field use can be ex-panded from food to social growth and biofuels as the world population increases. As aresourceful evolution, third-generation (algal) biofuels may avoid food competition andland use. The fast growth rate of algae enables the massive cultivation in non-arablelandmasses, thereby eliminating competition with land in use for crop production. Com-pared to conventional forests, agroecosystems, and other aquatic plants, microscopic algaehave higher growth rates and efficiency. Unlike other agricultural biodiesel feedstocks,it requires much less surface area [49]. For example, brown seaweeds produce 13.1 kgdry weight m−2 yr−1 compared to 10 kg dry weight m−2 yr−1 from sugarcane [102,103].Figure 12 highlights the land requirements of microalgae compared to other feedstock.


3.1. Land The primary goal of biofuel conservation is to conserve land. Field use can be ex-

panded from food to social growth and biofuels as the world population increases. As a resourceful evolution, third-generation (algal) biofuels may avoid food competition and land use. The fast growth rate of algae enables the massive cultivation in non-arable land-masses, thereby eliminating competition with land in use for crop production. Compared to conventional forests, agroecosystems, and other aquatic plants, microscopic algae have higher growth rates and efficiency. Unlike other agricultural biodiesel feedstocks, it re-quires much less surface area [49]. For example, brown seaweeds produce 13.1 kg dry weight m−2 yr−1 compared to 10 kg dry weight m−2 yr−1 from sugarcane [102,103]. Figure 12 highlights the land requirements of microalgae compared to other feedstock.

Figure 12. Land demand of microalgae oil compared to different biomass [104].

In addition, due to the limited dependency on farmland (compared to crop-based biofuels), algae lead to less habitat destruction. Consequently, by using microalgae as bi-odiesel feedstock, competition for agricultural land, particularly for human consumption, is significantly reduced [105]. Oil yields from microalgae can surpass those from oil plants such as rapeseed, palm, or sunflower per hectare as shown in Figure 13.

2.5

819

4097

10932

5121

0 2000 4000 6000 8000 10000 12000

Algae (50% TAG)

Oil palm

Sunflower

Soybean

Rapeseed

Area to produce global oil demand (hectares × 106)

Figure 12. Land demand of microalgae oil compared to different biomass [104].

In addition, due to the limited dependency on farmland (compared to crop-basedbiofuels), algae lead to less habitat destruction. Consequently, by using microalgae asbiodiesel feedstock, competition for agricultural land, particularly for human consumption,is significantly reduced [105]. Oil yields from microalgae can surpass those from oil plantssuch as rapeseed, palm, or sunflower per hectare as shown in Figure 13.

Sustainability 2021, 13, 12374 17 of 30Sustainability 2021, 13, x FOR PEER REVIEW 18 of 32

Figure 13. Comparison of microalgae oil yield (L oil/ha/yr) with other biodiesel feedstock [105].

In Malaysia, coastal areas and underutilized rice land are promising sites for massive microalgae cultivation [106]. Due to saltwater penetration, these lands are unproductive and therefore can be used to produce marine microalgae appropriate for saltwater [107,108]. The ‘Submariner’ research team has explored the possibilities of connecting both macro-and micro-algae development facilities to use an operational offshore wind farm in the Baltic Sea to reduce the burden on land availability [109]. The DOE estimates that if algae fuel replaced all the petroleum fuel in the United States, it would require only 15,000 square miles, which is a few thousand square miles larger than Maryland. This is less than one-seventh of the area devoted to corn production in the United States in 2000 [70]. Wigmosta et al. [110] examined the land, water, and resource availability in the United States and determined that about 43,107 hectares of land were suitable for algae culture in open ponds. This corresponds to a possible yearly output of 2.20 1011 L of algal oil, which is equivalent to 48% of the United States’ annual petroleum imports.

3.2. Water Water use concern is the main drawback associated with first-generation and second-

generation biofuels. Water is a limited resource, and a lack of it can severely affect well-being. In addition, current water problems are predicted to be intensified by climate change. As a result of the lack of freshwater sources worldwide and the inefficient usage of freshwater aquifers, only brackish water or seawater can be considered in broader ap-plication. The water footprint of a biofuel refers to the total volume of surface water needed for its production. Three types of green, blue, and gray algae are commonly con-sidered for the water footprint of biofuel production [111]. Footprints in green and blue water refer to evaporation during the period of processing. The footprint of graywater applies to the water ultimately released as waste. The water footprint of microalgae and terrestrial plants was examined by Zhang et al. [112]. It was found that the green water footprint for microalgae processing was about one-quarter of the average green water footprint for three plant species. Microalgae biodiesel has a WF of about 3726 kg water/kg biodiesel. Still, it is possible to recycle about 84% of this water, taking the WF down to 591

Figure 13. Comparison of microalgae oil yield (L oil/ha/yr) with other biodiesel feedstock [105].

In Malaysia, coastal areas and underutilized rice land are promising sites for massivemicroalgae cultivation [106]. Due to saltwater penetration, these lands are unproductiveand therefore can be used to produce marine microalgae appropriate for saltwater [107,108].The ‘Submariner’ research team has explored the possibilities of connecting both macro-and micro-algae development facilities to use an operational offshore wind farm in theBaltic Sea to reduce the burden on land availability [109]. The DOE estimates that ifalgae fuel replaced all the petroleum fuel in the United States, it would require only15,000 square miles, which is a few thousand square miles larger than Maryland. Thisis less than one-seventh of the area devoted to corn production in the United States in2000 [70]. Wigmosta et al. [110] examined the land, water, and resource availability in theUnited States and determined that about 43,107 hectares of land were suitable for algaeculture in open ponds. This corresponds to a possible yearly output of 2.20 1011 L of algaloil, which is equivalent to 48% of the United States’ annual petroleum imports.

3.2. Water

Water use concern is the main drawback associated with first-generation and second-generation biofuels. Water is a limited resource, and a lack of it can severely affect well-being. In addition, current water problems are predicted to be intensified by climate change.As a result of the lack of freshwater sources worldwide and the inefficient usage of fresh-water aquifers, only brackish water or seawater can be considered in broader application.The water footprint of a biofuel refers to the total volume of surface water needed forits production. Three types of green, blue, and gray algae are commonly considered forthe water footprint of biofuel production [111]. Footprints in green and blue water referto evaporation during the period of processing. The footprint of graywater applies tothe water ultimately released as waste. The water footprint of microalgae and terrestrialplants was examined by Zhang et al. [112]. It was found that the green water footprint formicroalgae processing was about one-quarter of the average green water footprint for threeplant species. Microalgae biodiesel has a WF of about 3726 kg water/kg biodiesel. Still,it is possible to recycle about 84% of this water, taking the WF down to 591 kg water/kgbiodiesel [113]. By using nutrients in wastewater and seawater, using algae reduces the


need for fresh water. Furthermore, by recycling and reusing the discharged water from theharvest process, up to 90.2% of the usage of topically discharged water can be restored to themanufacturing process [114]. Table 12 highlights the water footprint of biofuel feedstocks.

Table 12. A comparison of the blue–green water footprints of microalgae biofuel and other feedstocks.

Biofuel’s Feedstock Type of WaterFootprint

WaterFootprint Biofuel’s Feedstock

Sugar cane (Bioethanol) Blue + Green 139 [115]

Rapeseed (Biodiesel) Blue + Green 165 [116]

Microalgae (Biodiesel) Blue + Green 14–87 [117]

3.3. Energy

The net energy ratio is the ratio of the energy of algal biofuel to the energy invested inalgal production. Micro-algae have an energy content of 5–8 kWh/kg (18,000–28,800 kJ/kg)of dry weight [118]. The development of algal biodiesel could be feasible if the energyneeded to generate the microscopic algae and the energy necessary to turn the microscopicalgae into operational fuel is lower than that sum. As a result, the Net Energy Ratio can bewritten as

ENER =EOutEIn

=Energy in Algal Bio f uel

Energy Invested.

Microalgae are solar-powered cell factories that turn carbon dioxide into potential bio-fuels [119]. Microalgae are a quickly evolving photosynthetic species capable of converting9–10% of solar energy (average sunlight irradiance) into biomass, with a potential yield ofaround 77 g/biomass/m/day, which is about 280 tons per hectare per year [120,121]. In ahighly efficient seaweed processing method, prices for energy return on investment fromseaweed (0.44 to 1.37) for fermentation and ethanol distillation could be equivalent to corn(1.07) [122].

3.4. Socio-Economic Aspects

Jobs and profits, food security, economic progress, sustainable energy, economic via-bility, health and security, public acceptance, and equality of opportunity were defined associo-economic measures. Both jobs and local revenue are vital factors of development inalgal energy generation. To meet the challenge of long-term viability, the development ofsafer and more sustainable biofuels must be planned with third-generation biofuels. Thegrowing of macroalgae is relatively easy; the crop can be harvested in around 6 weeks withmodest initial capital expenditure. These features provide women with a significant oppor-tunity to generate capital for themselves and their families [123]. About 116,000 households,representing over one million people, planted over 58,000 hectares of seaweed in the Philip-pines. It is worth noting that more than half of the seaweed-harvesting population isunskilled or semi-skilled [124,125]. Wild seaweed harvesting is a vital part of the historyand practice of many nations. Women form a large proportion of the harvester workers inBrazil (assumed to be about 80%), and nori (Pyropia spp.) collection is typically performedby women in Japan.

Similarly, the bulk of seaweed harvesters in South Africa are women [126] whoseannual average income was quoted as US $5000; therefore, microalgae biofuel production,as a long-term sustainable sector, can also create opportunities for job development atall skill levels, close to traditional biofuels [127], and it also can be a safer choice whenpaired with existing complementary industries. The correlation regarding waste effluentbio-fixation and the production of usable co-products (e.g., feed, fertilizer) [128] may beeconomically advantageous to native communities in parallel, supplementing seasonalindustry profits. Algae (seaweeds and microalgae) is recognized by the European Commis-sion (2012, 2016) as such a good food safety choice that by 2054, the combined cultivationof algae could achieve 56 million metric tons of protein production, accounting for 18%


of the worldwide alternative protein industry [129,130]. Many algal biofuel companieshave pilot plant job figures that can be registered. Wholesalers of algal biofuels productsand technology, such as nutrients, CO2, polyethylene liners, PBRs, pumps, and workersfrom plants that have mutual storage services (e.g., CO2, nutrients) to biofuel facilities,are examples of indirect jobs [131]. Gallagher claims that [132] the economic viability ofmicroalgae biofuel development seems reasonable and relies on government support andpotential oil price. The list of sustainable indicators are presented in Table 13.

Table 13. List of sustainable development metrics for bioenergy with a focus on terrestrial feedstocks [133].

Category Indicator Sustainable System Design Goals

Social welfareOccupation

Wealth of householdsSecurity of foods

High wages and more job opportunitiesHigh-paying jobs and reducing fuel prices, raising household income

Algal biofuel using non-arable land and opportunities for food co-products

Energy Premium for energy securityVolatility in the price of petrol

Maximize the advantages of substituting algal biofuel for fossil fuel for oilsecurity dollars.

Reduce uncertainty of fuel prices.

External trade Terms of commerceVolume of commerce

Build situations such that fewer capital leaves a government agency to buy crude.Minimizing net fuel imports

Profitability Investment return (ROI)Net present value (NPV)

Build a constructive ROIBuild a positive NPV

Resourceconservation

Non-renewable energy resources depletionFossil fuel energy return on investment

(fossil EROI)

Reduce the dependency on fossil fuelsRaise fossil fuel EROI above 1 and finally above 3

Socialacceptability

Public opinionTransparency

Efficient engagement from stakeholdersRisk of catastrophe

Show a promising estimation of a high percentageDisplay a steadily rising or high value

Maintain catastrophe level at current occurrence or based on comparable technologies

3.5. Environmental Aspects

The sustainable development focuses on eliminating GHG pollution from the atmo-sphere, reducing human health by using renewable energy sources such as biofuels andextracting toxic pollutants from the environment. Water contamination and freshwaterretention shortages, combined with global warming, are now overwhelming global fearsand threatening biodiversity. The third-generation biofuels offer valuable insight into theclean energy approach and long-term sustainability. Algae is one of the most efficientbiological mechanisms for transforming sunlight into energy to absorb and convert carbondioxide to biomass. Around half of the global carbon fixation is carried out by algae [134].Ecological science has shown that the combination of the production of macroalgae withe.g., shrimp [103] and salmon [104], will remedy coastal eutrophication. Algal-basedwastewater treatment offers an efficient and cost-effective tool to eliminate organic andinorganic contaminant wastes from wastewater. The wastewater from these sources can bedivided into organic and inorganic compounds. The critical portion of organic waste iscarbon-containing biodegradable substances. In contrast, inorganic waste includes nitrates,phosphates, and heavy metals [135]; hence, it provides a reliable and cost-effective toolto extract wastewater from organic and inorganic toxins. The author of [122] has recentlyshown how the rare metal indium could be extracted from e-waste (old electronic materials)that is frequently transported to developing nations, where workers (many of whom areminors) burn circuit boards to remove valuable and rare metals. This has grave health andpollution consequences. The brown seaweed Ascophyllum nodosum biomass was discoveredto aid in the ‘extraction’ of the metal indium, which is essential as an input to modern elec-tronics, particularly screens. Biofuels derived from microscopic algae are one of the mostpromising green energy options not just because of their reduced greenhouse gas emissionsbut also because of their CO2 sequestration [136]. The CO2 sequestration in microalgaeis 10–50 times more than that of many terrestrial plants, while a higher concentration ofCO2 results in a higher yield of lipids. In contrast, fourth-generation biofuel is pushed to


be carbon-negative with significant environmental benefits. Table 14 highlights the GHGreduction of genetically engineered microalgae.

Table 14. Fixation of greenhouse gases by genetically engineered microalgae.

Species Method Attainment References

Chlamydomonas reinhardtii

Engineered small PSII antenna size Chlamydomonas reinhardtii bytransforming a permanently active variant NAB1*

(mutagenized NAB1) of the LHC translation repressor NAB1 todecrease antenna size by translation repression.

Photosynthesis efficiencyhas increased by 50%. [137]

Synechococcus elongatus(Strain of freshwater

cyanobacteria).

Genetically modified cyanobacteria generate and secrete carbonicanhydrase (Cas) in the medium. The secreted CAs converted

dissolved CO2 to HCO3. The cyanobacteria absorbed HCO3 andconverted it into biomass through photosynthesis.

Carbon intake hasincreased by 41%. [138]

3.6. Current and Future Prospects of Biofuels

In 2019, global production of biofuels increased 5%, which was led mainly by a 13%biodiesel expansion (with Indonesia overtaking the US and Brazil to develop into thesignificant national producer (Figure 14). Meanwhile, bioethanol production increased by2%. In 2019, global biofuel jobs were projected at 2.5 million [139].


wastewater treatment offers an efficient and cost-effective tool to eliminate organic and inorganic contaminant wastes from wastewater. The wastewater from these sources can be divided into organic and inorganic compounds. The critical portion of organic waste is carbon-containing biodegradable substances. In contrast, inorganic waste includes ni-trates, phosphates, and heavy metals [135]; hence, it provides a reliable and cost-effective tool to extract wastewater from organic and inorganic toxins. The author of [122] has re-cently shown how the rare metal indium could be extracted from e-waste (old electronic materials) that is frequently transported to developing nations, where workers (many of whom are minors) burn circuit boards to remove valuable and rare metals. This has grave health and pollution consequences. The brown seaweed Ascophyllum nodosum biomass was discovered to aid in the ‘extraction’ of the metal indium, which is essential as an input to modern electronics, particularly screens. Biofuels derived from microscopic algae are one of the most promising green energy options not just because of their reduced green-house gas emissions but also because of their CO2 sequestration [136]. The CO2 sequestra-tion in microalgae is 10–50 times more than that of many terrestrial plants, while a higher concentration of CO2 results in a higher yield of lipids. In contrast, fourth-generation bio-fuel is pushed to be carbon-negative with significant environmental benefits. Table 14 highlights the GHG reduction of genetically engineered microalgae

Table 14. Fixation of greenhouse gases by genetically engineered microalgae.

Species Method Attainment References

Chlamydomonas reinhardtii

Engineered small PSII antenna size Chlamydomonas reinhardtii by transforming a permanently active variant NAB1* (mutagenized NAB1) of the LHC translation re-

pressor NAB1 to decrease antenna size by translation re-pression.

Photosynthe-sis efficiency has increased

by 50%.

[137]

Synechococcus elongatus (Strain of freshwater

cyanobacteria).

Genetically modified cyanobacteria generate and secrete carbonic anhydrase (Cas) in the medium. The secreted CAs

converted dissolved CO2 to HCO3. The cyanobacteria absorbed HCO3 and converted it into biomass through

photosynthesis.

Carbon intake has increased

by 41%. [138]

3.6. Current and Future Prospects of Biofuels In 2019, global production of biofuels increased 5%, which was led mainly by a 13%

biodiesel expansion (with Indonesia overtaking the US and Brazil to develop into the sig-nificant national producer(Figure 14). Meanwhile, bioethanol production increased by 2%. In 2019, global biofuel jobs were projected at 2.5 million [139].

0.000.200.400.600.801.00

JOBS

(mill

ions

)

2020 2019

Figure 14. Liquid biofuel employment in top 10 countries [139,140].

According to IEA, 3% annual production growth is projected for the next five years, butthe decline in oil prices in 2020 (USD 30 per barrel) due to lower global demand stemmingprimarily from the COVID-19 pandemic decreased demand for biofuel crops [141]. Liquidbiofuels are believed to be one of the most cost-competitive suppliers of high efficiency anda potential substitute for marine and aircraft fuels. The biofuel market was heavily affectedby the COVID-19 pandemic in 2020. Global biofuel transport production is expected to be144 billion liters in 2020, which is equivalent to 2,480,000 barrels per day (kb/d): an 11.6%decrease from peak production in 2019 and the first decline in annual production in severaldecades [139]. Figure 15 depicts the increase in yearly biofuel demand in various countriesto meet the 2030 sustainable development scenario. Biofuel production in the United Statesand EU member states will fall short of SDS demand in 2030. While biofuel productionin Brazil and India is estimated to rise, the SDS volume for 2030 must involve even fastergrowth. China and ASEAN countries are also experiencing production growth, which, ifmaintained, would meet the SDS’s 2030 biofuel volume requirements [142].



Figure 14. Liquid biofuel employment in top 10 countries [139,140].

According to IEA, 3% annual production growth is projected for the next five years, but the decline in oil prices in 2020 (USD 30 per barrel) due to lower global demand stem-ming primarily from the COVID-19 pandemic decreased demand for biofuel crops [141]. Liquid biofuels are believed to be one of the most cost-competitive suppliers of high effi-ciency and a potential substitute for marine and aircraft fuels. The biofuel market was heavily affected by the COVID-19 pandemic in 2020. Global biofuel transport production is expected to be 144 billion liters in 2020, which is equivalent to 2,480,000 barrels per day (kb/d): an 11.6% decrease from peak production in 2019 and the first decline in annual production in several decades [139]. Figure 15 depicts the increase in yearly biofuel de-mand in various countries to meet the 2030 sustainable development scenario. Biofuel production in the United States and EU member states will fall short of SDS demand in 2030. While biofuel production in Brazil and India is estimated to rise, the SDS volume for 2030 must involve even faster growth. China and ASEAN countries are also experiencing production growth, which, if maintained, would meet the SDS’s 2030 biofuel volume re-quirements [142].

Figure 15. Biofuels annual production growth to meet sustainable development scenario, 2030 [142].

4. Transition to a Circular Economy, Green Economy, and Bioeconomy 4.1. Circular Economy

The fundamental principle that connects the ideas of circular economy, green econ-omy, and bioeconomy is balancing economic, environmental, and social objectives. The circular economy is a possible solution to the optimal utilization of investments and en-sures their long-term use. The processing units must demonstrate economic feasibility while minimizing waste and environmental effects to achieve a fully integrated circular bioeconomy. The new green deal from the European Commission focuses on priority ar-eas where algae production may make a significant contribution: for example, the goals of the EU becoming climate neutral by 2050, the protection of biodiversity [143], and the development of a circular economy [144]. The circular economy is based on three basic principles: a. No waste, since products are renewable and biodegradable. b. Consumed resources are recovered without posing any security threats to the eco-

system. c. Energy for all processes is provided from renewable and sustainable sources.

1.9% 0.5% 1.7%

11.8%15.3%

13.3%

7%9%

5%

22%19%

10%

0.0%

5.0%

10.0%

15.0%

20.0%

25.0%

United States

European Union

Brazil India China ASEAN

Forecast annual production growth (2019-25)

Annual production growth needed to meet SDS (2019-30)

Figure 15. Biofuels annual production growth to meet sustainable development scenario, 2030 [142].

4. Transition to a Circular Economy, Green Economy, and Bioeconomy4.1. Circular Economy

The fundamental principle that connects the ideas of circular economy, green economy,and bioeconomy is balancing economic, environmental, and social objectives. The circulareconomy is a possible solution to the optimal utilization of investments and ensures theirlong-term use. The processing units must demonstrate economic feasibility while mini-mizing waste and environmental effects to achieve a fully integrated circular bioeconomy.The new green deal from the European Commission focuses on priority areas where algaeproduction may make a significant contribution: for example, the goals of the EU becomingclimate neutral by 2050, the protection of biodiversity [143], and the development of acircular economy [144]. The circular economy is based on three basic principles:

a. No waste, since products are renewable and biodegradable.b. Consumed resources are recovered without posing any security threats to the ecosystem.c. Energy for all processes is provided from renewable and sustainable sources.

Vitamins, proteins, amino acids, polysaccharides, fatty acids, sterols, pigments, fibers,and enzymes with unique properties can be synthesized from microalgae. In a microalgae-based circular bioeconomy, production wastes are recycled and reintroduced as secondaryraw materials, i.e., to convert waste materials into new products in microalgae-based pro-duction systems (microalgae biorefineries). Microalgae are helpful in a circular economyas they can be used for the bio-remediation of nutrient waste and provide biomass forvarious commercial uses. Microalgal farming on nonarable land or coastal ecosystemsreduces water demands, recycles nutrients, and converts atmospheric CO2 into nutrient-rich sustainable feedstocks. This lays the groundwork for a circular aquaculture-basedindustry as part of a larger circular bioeconomy [145] contributing to several UN Sustain-able Development Goals. The circular bioeconomy principle is currently gaining attentionas a critical component of green technology. Recently, combined activated sludge (AS)microalgae wastewater treatment systems have been suggested as a more energy and com-mercially efficient alternative to traditional solutions for removing carbon and nutrientsfrom liquid streams.

Furthermore, microalgae cultivation in wastewater leads to faster nitrogen and phos-phorus removal, with up to 1 kg of dry biomass generated per m3 of wastewater [146]. TheEU goals for creating circular economy from waste sources [147] align with current urbanwater management paradigms [148]. Bioplastics are critical in transitioning the plasticssector from a wasteful linear economy to a circular economy. Algae-based bioplastic isconsidered to be a long-term solution for ensuring the circular economy practice. Bioplasticcould produce natural materials via composting as the end of life cycle management [149].


Microalgae are a viable alternative source for making bioplastics. Several recent stud-ies have looked at the production of bioplastics from microalgae biomass. According toKaran et al. (2019), the average requirement for microalgae cultivation to meet globalplastic manufacturing is about 145 000 km2, which is only 0.028% of the Earth’s surface areaof 510,000,000 km2 [150]. Polysaccharides agar, carrageenan, and alginate are used to makebioplastics from seaweeds, and seaweed waste from agar extraction has been suggested asa material filler [151].The methods of producing PHA from genetically engineering algae ispresented in Table 15.

Table 15. Methods of producing PHAs from genetically engineered algae.

Algae Type of Product Culture Mode Polymer(Percentage of Dry Cell Weight) Reference

Spirulina plantesis PHB Production of P(3HB) usingCO2/acetate as a carbon source. 10 [152]

Nostoc muscorum PHB

P(3HB) production underphosphate-starved medium + 1%(w/w) glucose + 1% (w/w) acetatewith aeration and CO2 addition.

21.5 [153]

A circular economy-based business model for obtaining several products from mi-croalgae biomass for agricultural, nutrition, cosmetics, and aquaculture use is proposed ina study [154]. AlgaePro is developing technologies for growing microalgae in a circulareconomy approach, using biodegradables from urban waste, CO2, and waste heat fromindustrial sites [155]. Researchers in Italy and Slovenia are cultivating microalgae thatabsorb nutrients from agricultural wastewater as part of a European initiative called Salt-gae. Once the water has been cleaned, the algae are dried and sold in cosmetics, animalfeed, and fertilizers. Aquaculture of algae on industrial sites would enable a circulareconomy, turning wastewater into a viable resource [156].A circular based economy usingMicro/Macroalgae is presented in Figure 16.


Figure 16. Systematic diagram of a circular economy.

4.2. Green Economy The green economy is a viable alternative to today’s economic framework, which ag-

gravates inequality, stimulates pollution, induces resource scarcity, and poses numerous environmental health risks. According to the UNEP [157] (, a green economy is “low-car-bon, resource-effective and socially equitable”, with the ultimate goal of reducing envi-ronmental impact and biodiversity loss as well as improving human well-being and social justice. The numerous benefits associated with algal energy, such as eco-friendliness and high productivity, lead to a green economy and sustainable growth by improving human health and quality of life [157].The benefits of green economy using algae is presented in Figure 17.

4.3. Bioeconomy Rapid urbanization, improved quality of life, and longer lifespans place demands on

all manufacturing sectors producing food, chemicals, and fuels. As a result of the in-creased strain, land usage, drinkable water, fossil fuels, and other natural resources are anticipated to increase, resulting in unexpected climate change, biodiversity loss, and a decline in the capacity to manage ecosystems sustainably. The bioeconomy may offer a potential solution to this rising demand by substituting biomass-based commodities for depletable resources, reducing environmental impact. A bioeconomy is defined as “the development of long-term biological resources and the conversion of waste biological re-sources into value-added products such as food, feed, bio-based products, and bioenergy” [158]. The European Union introduced a plan for improving the bioeconomy in 2014, which was based on microalgae. Microalgae can significantly contribute to the economy, providing required biomass for human applications such as new drugs, cosmetics, food, and feed. The plan also included options for wastewater treatment and atmospheric CO2 mitigation. Increasing the market development for microalgae-based products as long-term substitutes for currently available options will be critical to the success of a microal-gae-based bioeconomy. The industrial units of the bioeconomy are biorefineries. The enor-mous potential of tiny microalgae favors a microalgae-based biorefinery and bioeconomy, generating huge opportunities in the global algae industry. Seaweeds can also be used as feedstock in biorefineries to produce fuels, pesticides, food additives, medicines, and other products, making them an essential part of the future bioeconomy [159].

Figure 16. Systematic diagram of a circular economy.

4.2. Green Economy

The green economy is a viable alternative to today’s economic framework, which ag-gravates inequality, stimulates pollution, induces resource scarcity, and poses numerousenvironmental health risks. According to the UNEP [157] (a green economy is “low-carbon,resource-effective and socially equitable”, with the ultimate goal of reducing environmental


impact and biodiversity loss as well as improving human well-being and social justice. Thenumerous benefits associated with algal energy, such as eco-friendliness and high productivity,lead to a green economy and sustainable growth by improving human health and quality oflife [157].The benefits of green economy using algae is presented in Figure 17.


Figure 17. Algal-based circular economy, green economy, and bioeconomy.

5. Conclusions Many developed and developing nations are steadily supporting biofuel production

due to its potential benefits. This study examined the prospects of third-generation and fourth-generation biofuels in the context of long-term sustainability. The following are some of the key conclusions from the study. • Greenhouse gas emissions, environmental impact, loss of habitat, community con-

flicts, and substantial production costs are all associated with first-generation and second-generation biofuel. The use of edible biomass in first-generation biofuels has been of significant concern. It competes with the world’s food requirements that limit its production to a few countries. The other limitation includes the high investment costs and poor efficiencies of feedstock conversion to biofuel. Second-generation bio-fuel has production limitations. Both the first and second generations have their strengths and weaknesses in terms of environmental and social impact. Hence, both generations will shortly be unable to meet the growing biofuel demand and energy transition targets.

• Developing third-generation and fourth-generation biofuels has broad implications on global socio-economic growth and sustainable development goals. It contributes to carbon balance, biodiversity conservation, sustainable water utilization, healthy air quality, soil conservation, and sustainable social enterprise.

• A large number of companies are investing heavily in biofuels to accelerate the global energy transition. Creating and applying sustainable biofuels standards will be more critical, with more entrepreneurs or companies committed to thinking that benefits will ultimately outweigh the risks.

• Nanotechnology has the potential to make next-generation biofuels feasible. The ef-ficacy of biofuel can be significantly enhanced by incorporating nanomaterials into the process development. Magnetic nanoparticles, carbon nanotubes, metal oxide na-noparticles, and other Nano catalysts have the potential to become an essential part of long-term bioenergy production. However, most of the performance data are based on small-scale biofuel generation. Further research is needed to study the effi-cacy of nanotechnology in pilot-scale biofuel production.

Figure 17. Algal-based circular economy, green economy, and bioeconomy.

4.3. Bioeconomy

Rapid urbanization, improved quality of life, and longer lifespans place demands onall manufacturing sectors producing food, chemicals, and fuels. As a result of the increasedstrain, land usage, drinkable water, fossil fuels, and other natural resources are anticipatedto increase, resulting in unexpected climate change, biodiversity loss, and a decline in thecapacity to manage ecosystems sustainably. The bioeconomy may offer a potential solutionto this rising demand by substituting biomass-based commodities for depletable resources,reducing environmental impact. A bioeconomy is defined as “the development of long-term biological resources and the conversion of waste biological resources into value-addedproducts such as food, feed, bio-based products, and bioenergy” [158]. The European Unionintroduced a plan for improving the bioeconomy in 2014, which was based on microalgae.Microalgae can significantly contribute to the economy, providing required biomass forhuman applications such as new drugs, cosmetics, food, and feed. The plan also includedoptions for wastewater treatment and atmospheric CO2 mitigation. Increasing the marketdevelopment for microalgae-based products as long-term substitutes for currently availableoptions will be critical to the success of a microalgae-based bioeconomy. The industrialunits of the bioeconomy are biorefineries. The enormous potential of tiny microalgae favorsa microalgae-based biorefinery and bioeconomy, generating huge opportunities in theglobal algae industry. Seaweeds can also be used as feedstock in biorefineries to producefuels, pesticides, food additives, medicines, and other products, making them an essentialpart of the future bioeconomy [159].

5. Conclusions

Many developed and developing nations are steadily supporting biofuel productiondue to its potential benefits. This study examined the prospects of third-generation and


fourth-generation biofuels in the context of long-term sustainability. The following aresome of the key conclusions from the study.

• Greenhouse gas emissions, environmental impact, loss of habitat, community conflicts,and substantial production costs are all associated with first-generation and second-generation biofuel. The use of edible biomass in first-generation biofuels has beenof significant concern. It competes with the world’s food requirements that limit itsproduction to a few countries. The other limitation includes the high investment costsand poor efficiencies of feedstock conversion to biofuel. Second-generation biofuel hasproduction limitations. Both the first and second generations have their strengths andweaknesses in terms of environmental and social impact. Hence, both generations willshortly be unable to meet the growing biofuel demand and energy transition targets.

• Developing third-generation and fourth-generation biofuels has broad implicationson global socio-economic growth and sustainable development goals. It contributes tocarbon balance, biodiversity conservation, sustainable water utilization, healthy airquality, soil conservation, and sustainable social enterprise.

• A large number of companies are investing heavily in biofuels to accelerate the globalenergy transition. Creating and applying sustainable biofuels standards will be morecritical, with more entrepreneurs or companies committed to thinking that benefitswill ultimately outweigh the risks.

• Nanotechnology has the potential to make next-generation biofuels feasible. Theefficacy of biofuel can be significantly enhanced by incorporating nanomaterials intothe process development. Magnetic nanoparticles, carbon nanotubes, metal oxidenanoparticles, and other Nano catalysts have the potential to become an essential partof long-term bioenergy production. However, most of the performance data are basedon small-scale biofuel generation. Further research is needed to study the efficacy ofnanotechnology in pilot-scale biofuel production.

• Biofuels from macroalgae and microalgae contribute to a circular economy by gen-erating natural bio-products, such as proteins, pigments, fatty acids, and bioplastics.Therefore, algae-based green and bioeconomy opportunities include a new supplychain in manufacturing, cleaner fuel, food security, and GHG mitigation benefits.

• With the advancement of technology, extensive research, and development, it isreasonable to assume that third-generation and fourth-generation biofuel will becomemore appealing for commercial usage globally.

• If specific considerations related to sustainability requirements are met, third-generationand fourth-generations biofuels can be used realistically as a transitional approach.In the future, nature-inspired solutions hold more excellent prospects as sustainableenergy sources for the planet.

Author Contributions: Conceptualization, K.S. and R.M.; investigation, K.S.; resources, R.M.; datacuration, N.K.; writing—original draft preparation, N.K.; writing—review and editing, K.S.; visu-alization, N.K.; supervision, K.S., R.M.; project administration, K.S.; funding acquisition, K.S. Allauthors have read and agreed to the published version of the manuscript.

Funding: The authors are grateful for the financial assistance granted by the Universiti MalaysiaPahang (www.ump.edu.my accessed on 25 October 2021) under the Postgraduate Research GrantsScheme (PGRS) PGRS210348.

Conflicts of Interest: The authors declare no conflict of interest.

www.ump.edu.my


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